Deepa Sridharan

Nonerythroid alphaII spectrin is required for recruitment of FANCA and XPF to nuclear foci induced by DNA interstrand cross-links.

The events responsible for repair of DNA interstrand cross-links in mammalian cells, the proteins involved and their interactions with each other are poorly understood. The present study demonstrates that the structural protein nonerythroid α spectrin (αSpIIΣ*), present in normal human cell nuclei, plays an important role in repair of DNA interstrand cross-links. These results show that αSpIIΣ* relocalizes to nuclear foci after damage of normal human cells with the DNA interstrand cross-linking agent 8-methoxypsoralen plus ultraviolet A (UVA) light and that FANCA and the known DNA repair protein XPF localize to the same nuclear foci. That αSpIIΣ* is essential for this re-localization is demonstrated by the finding that in cells from patients with Fanconi anemia complementation group A (FA-A), which have decreased ability to repair DNA interstrand cross-links and decreased levels of αSpIIΣ*, there is a significant reduction in formation of damage-induced XPF as well asα SpIIΣ* nuclear foci, even though levels of XPF are normal in these cells. In corrected FA-A cells, in which levels of αSpIIΣ* are restored to normal, numbers of damage-induced nuclear foci are also returned to normal. Co-immunoprecipitation studies show thatα SpIIΣ*, FANCA and XPF co-immunoprecipitate with each other from normal human nuclear proteins. These results demonstrate thatα SpIIΣ*, FANCA and XPF interact with each other in the nucleus and indicate that there is a close functional relationship between these proteins. These studies suggest that an important role for αSpIIΣ* in the nucleus is to act as a scaffold, aiding in recruitment and alignment of repair proteins at sites of damage.

Understanding cancer development processes after HZE-particle exposure: roles of ROS, DNA damage repair and inflammation

During space travel astronauts are exposed to a variety of radiations, including galactic cosmic rays composed of high-energy protons and high-energy charged (HZE) nuclei, and solar particle events containing low- to medium-energy protons. Risks from these exposures include carcinogenesis, central nervous system damage and degenerative tissue effects. Currently, career radiation limits are based on estimates of fatal cancer risks calculated using a model that incorporates human epidemiological data from exposed populations, estimates of relative biological effectiveness and dose-response data from relevant mammalian experimental models. A major goal of space radiation risk assessment is to link mechanistic data from biological studies at NASA Space Radiation Laboratory and other particle accelerators with risk models. Early phenotypes of HZE exposure, such as the induction of reactive oxygen species, DNA damage signaling and inflammation, are sensitive to HZE damage complexity. This review summarizes our current understanding of critical areas within the DNA damage and oxidative stress arena and provides insight into their mechanistic interdependence and their usefulness in accurately modeling cancer and other risks in astronauts exposed to space radiation. Our ultimate goals are to examine potential links and crosstalk between early response modules activated by charged particle exposure, to identify critical areas that require further research and to use these data to reduced uncertainties in modeling cancer risk for astronauts. A clearer understanding of the links between early mechanistic aspects of high-LET response and later surrogate cancer end points could reveal key nodes that can be therapeutically targeted to mitigate the health effects from charged particle exposures.

Knockdown of μ-calpain in Fanconi Anemia, FA-A, cells by siRNA Restores αII Spectrin levels and Corrects Chromosomal Instability and Defective DNA Interstrand Cross-link Repair

We have previously shown that there is a deficiency in the structural protein, nonerythroid alpha spectrin (alphaIISp), in cells from patients with Fanconi anemia (FA). These studies indicate that this deficiency is due to the reduced stability of alphaIISp and correlates with a decreased level of repair of DNA interstrand cross-links and chromosomal instability in FA cells. An important factor in the stability of alphaIISp is its susceptibility to cleavage by the protease, mu-calpain. We hypothesized that an increased level of mu-calpain cleavage of alphaIISp in FA cells leads to an increased level of breakdown of alphaIISp and that knocking down expression of mu-calpain in FA cells should restore levels of alphaIISp and correct a number of the phenotypic defects observed. The results showed that there is increased mu-calpain activity in FA-A, FA-C, FA-D2, FA-F, and FA-G cells that could account for the deficiency in alphaIISp in these FA cells. Protein interaction studies indicated that FANCA and FANCG bind directly to mu-calpain. We hypothesize that this binding may lead to inhibition of mu-calpain activity in normal cells. Knocking down mu-calpain by siRNA in FA-A cells restored levels of alphaIISp to normal and reversed a number of the cellular deficiencies in these cells. It corrected the DNA repair defect and the chromosomal instability observed after exposure to a DNA interstrand cross-linking agent. These studies indicate that FA proteins may play an important role in maintaining the stability of alphaIISp in the cell by regulating its cleavage by mu-calpain. Thus, by reducing the level of breakdown of alphaIISp in FA cells, we may be able to reverse a number of the cellular deficiencies observed in this disorder.

The SH3 Domain of αII Spectrin Is a Target for the Fanconi Anemia Protein, FANCG†

The structural protein nonerythroid alpha spectrin (alphaIISp) plays a role in the repair of DNA interstrand cross-links and is deficient in cells from patients with Fanconi anemia (FA), in which there is a defect in ability to repair such cross-links. We have proposed a model in which alphaIISp, whose stability is dependent on FA proteins, acts as a scaffold to aid in recruitment of repair proteins to sites of damage. In order to get a clearer understanding of the proposed role of FA proteins in maintaining stability of alphaIISp, yeast two-hybrid analysis was carried out to determine whether FA proteins directly interact with alphaIISp and, if so, to map the sites of interaction. Four overlapping regions of alphaIISp were constructed. FANCG interacted with one of these regions and specifically with the SH3 domain in this region of alphaIISp. The site of interaction in FANCG was mapped to a motif that binds to SH3 domains and contains a consensus sequence with preference for the SH3 domain of alphaIISp. This site of interaction was confirmed using site-directed mutagenesis. Two FA proteins that did not contain motifs that bind to SH3 domains, FANCC and FANCF, did not interact with the SH3 domain of alphaIISp. These results demonstrate that one of the FA proteins, FANCG, contains a motif that interacts directly with the SH3 domain of alphaIISp. We propose that this binding of FANCG to alphaIISp may be important for the stability of alphaIISp in cells and the role alphaIISp plays in the DNA repair process.

Defining the Biological Effectiveness of Components of High-LET Track Structure

During space travel, astronauts are exposed to a wide array of high-linear energy transfer (LET) particles, with differing energies and resulting biological effects. Risk assessment of these exposures carries a large uncertainty predominantly due to the unique track structure of the particles energy deposition. The complex damage elicited by high charge and energy (HZE) particles results from both lesions along the track core and from energetic electrons, δ rays, generated as a consequence of particle traversal. To better define how cells respond to this complex radiation exposure, a normal hTERT immortalized skin fibroblast cell line was exposed to a defined panel of particles carefully chosen to tease out track structure effects. Phosphorylation kinetics for several key double-strand break (DSB) response proteins (γ-H2AX, pATF2 and pSMC1) were defined after exposure to ten different high-LET radiation qualities and one low-LET radiation (X ray), at two doses (0.5–2 Gy) and time points (2 and 24 h). The results reveal that the lower energy particles (Fe 300, Si 93 and Ti 300 MeV/u), with a narrower track width and higher number and intensity of δ rays, cause the highest degree of persistent damage response. The persistent γ-H2AX signal at lower energies suggests that damage from these exposures are more difficult to resolve, likely due to the greater complexity of the associated DNA lesions. However, different kinetics were observed for the solely ATM-mediated phosphorylations (pATF2 and pSMC1), revealing a shallow induction at early times and a higher level of residual phosphorylation compared to γ-H2AX. The differing phospho-protein profiles exhibited, compared to γ-H2AX, suggests additional functions for these proteins within the cell. The strong correspondence between the predicted curves for energy deposition per nucleosome for each ion/energy combination and the persistent levels of γ-H2AX indicates that the nature of energy distribution defines residual levels of γ-H2AX, an indicator of unrepaired DSBs. Our results suggest that decreasing the energy of a particle results in more complex damage that may increase genomic instability and increase the risk of carcinogenesis.

Protons sensitize epithelial cells to mesenchymal transition

Proton radiotherapy has gained more favor among oncologists as a treatment option for localized and deep-seated tumors. In addition, protons are a major constituent of the space radiation astronauts receive during space flights. The potential for these exposures to lead to, or enhance cancer risk has not been well studied. Our objective is to study the biological effects of low energy protons on epithelial cells and its propensity to enhance transforming growth factor beta 1 (TGFβ1)-mediated epithelial-mesenchymal transition (EMT), a process occurring during tumor progression and critical for invasion and metastasis. Non-transformed mink lung epithelial cells (Mv1Lu) and hTERT- immortalized human esophageal epithelial cells (EPC) were used in this study. EMT was identified by alterations in cell morphology, EMT-related gene expression changes determined using real-time PCR, and EMT changes in specific cellular markers detected by immunostaining and western blotting. Although TGFβ1 treatment alone is able to induce EMT in both Mv1Lu and EPC cells, low energy protons (5 MeV) at doses as low as 0.1 Gy can enhance TGFβ1 induced EMT. Protons alone can also induce a mild induction of EMT. SD208, a potent TGFβ Receptor 1 (TGFβR1) kinase inhibitor, can efficiently block TGFβ1/Smad signaling and attenuate EMT induction. We suggest a model for EMT after proton irradiation in normal and cancerous tissue based on our results that showed that low and high doses of protons can sensitize normal human epithelial cells to mesenchymal transition, more prominently in the presence of TGFβ1, but also in the absence of TGFβ1.

Evaluating biomarkers to model cancer risk post cosmic ray exposure.

Robust predictive models are essential to manage the risk of radiation-induced carcinogenesis. Chronic exposure to cosmic rays in the context of the complex deep space environment may place astronauts at high cancer risk. To estimate this risk, it is critical to understand how radiation-induced cellular stress impacts cell fate decisions and how this in turn alters the risk of carcinogenesis. Exposure to the heavy ion component of cosmic rays triggers a multitude of cellular changes, depending on the rate of exposure, the type of damage incurred and individual susceptibility. Heterogeneity in dose, dose rate, radiation quality, energy and particle flux contribute to the complexity of risk assessment. To unravel the impact of each of these factors, it is critical to identify sensitive biomarkers that can serve as inputs for robust modeling of individual risk of cancer or other long-term health consequences of exposure. Limitations in sensitivity of biomarkers to dose and dose rate, and the complexity of longitudinal monitoring, are some of the factors that increase uncertainties in the output from risk prediction models. Here, we critically evaluate candidate early and late biomarkers of radiation exposure and discuss their usefulness in predicting cell fate decisions. Some of the biomarkers we have reviewed include complex clustered DNA damage, persistent DNA repair foci, reactive oxygen species, chromosome aberrations and inflammation. Other biomarkers discussed, often assayed for at longer points post exposure, include mutations, chromosome aberrations, reactive oxygen species and telomere length changes. We discuss the relationship of biomarkers to different potential cell fates, including proliferation, apoptosis, senescence, and loss of stemness, which can propagate genomic instability and alter tissue composition and the underlying mRNA signatures that contribute to cell fate decisions. Our goal is to highlight factors that are important in choosing biomarkers and to evaluate the potential for biomarkers to inform models of post exposure cancer risk. Because cellular stress response pathways to space radiation and environmental carcinogens share common nodes, biomarker-driven risk models may be broadly applicable for estimating risks for other carcinogens.

Increased Artemis levels confer radioresistance to both high and low LET radiation exposures

BackgroundArtemis has a defined role in V(D)J recombination and has been implicated in the repair of radiation induced double-strand breaks. However the exact function(s) of Artemis in DNA repair and its preferred substrate(s) in vivo remain undefined. Our previous work suggests that Artemis is important for the repair of complex DNA damage like that inflicted by high Linear Energy Transfer (LET) radiation. To establish the contribution of Artemis in repairing DNA damage caused by various radiation qualities, we evaluated the effect of over-expressing Artemis on cell survival, DNA repair, and cell cycle arrest after exposure to high and low LET radiation.ResultsOur data reveal that Artemis over-expression confers marked radioprotection against both types of radiation, although the radioprotective effect was greater following high LET radiation. Inhibitor studies reveal that the radioprotection imparted by Artemis is primarily dependent on DNA-PK activity, and to a lesser extent on ATM kinase activity. Together, these data suggest a DNA-PK dependent role for Artemis in the repair of complex DNA damage.ConclusionsThese findings indicate that Artemis levels significantly influence radiation toxicity in human cells and suggest that Artemis inhibition could be a practical target for adjuvant cancer therapies.

Genetic Variation and Radiation Quality Impact Cancer Promoting Cellular Phenotypes in Response to HZE exposure

There exists a wide degree of genetic variation within the normal human population which includes disease free individuals with heterozygote defects in major DNA repair genes. A lack of understanding of how this genetic variation impacts cellular phenotypes that inform cancer risk post heavy ion exposure poses a major limitation in developing personalized cancer risk assessment astronauts. We initiated a pilot study with Human Mammary Epithelial Cell strains (HMEC) derived from wild type, a p16 silenced derivative of wild type, and various genetic variants that were heterozygote for DNA repair genes; BRCA1, BRCA2 and ATM. Cells strains were exposed to different high and low LET radiation qualities to generate both simple and complex lesions and centrosome aberrations were examined as a surrogate marker of genomic instability and cancer susceptibility post different exposures. Our results indicate that centrosome aberration frequency is higher in the genetic variants under study. The aberration frequency increases with dose, complexity of the lesion generated by different radiation qualities and age of the individual. This increase in genomic instability correlates with elevated check-point activation post radiation exposure. These studies suggest that the influence of individual genetics on cell cycle regulation could modify the degree of early genomic instability in response to complex lesions and potentially define cancer predisposition in response to HZE exposure. These results will have significant implications in estimating cancer susceptibility in genetically variant individuals exposed to HZE particles.

Lesion complexity drives age related cancer susceptibility in human mammary epithelial cells

Exposures to various DNA damaging agents can deregulate a wide array of critical mechanisms that maintain genome integrity. It is unclear how these processes are impacted by ones age at the time of exposure and the complexity of the DNA lesion. To clarify this, we employed radiation as a tool to generate simple and complex lesions in normal primary human mammary epithelial cells derived from women of various ages. We hypothesized that genomic instability in the progeny of older cells exposed to complex damages will be exacerbated by age-associated deterioration in function and accentuate age-related cancer predisposition. Centrosome aberrations and changes in stem cell numbers were examined to assess cancer susceptibility. Our data show that the frequency of centrosome aberrations proportionately increases with age following complex damage causing exposures. However, a dose-dependent increase in stem cell numbers was independent of both age and the nature of the insult. Phospho-protein signatures provide mechanistic clues to signaling networks implicated in these effects. Together these studies suggest that complex damage can threaten the genome stability of the stem cell population in older people. Propagation of this instability is subject to influence by the microenvironment and will ultimately define cancer risk in the older population.